Name: comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods
Uploaded: 2018-09-10T14:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods at JoVE.com

이 작품에서 국립 자연 과학 재단 (81771617) 및 자연 과학 재단의 광 동 지방 (2016A030313178) X.F.;에 게 수 여 교부 금에 의해 지원 되었다 심천 기본 연구 기금 (JCYJ20170413165233512) X.F;에 수 여에서 부여 유 니스 케네디 슈 라이버 국립 연구소의 아동 건강 및 보너스 번호 R01HD088549에서 건강의 국가 학회의 인간 발달 (전적으로 저자의 책임은 내용과 반드시 공식 대표 하지 않는다 국립 보건원의 조회) N.N. 하

모든 마우스 실험 동물 관리 및 사용 위원회의 심천 연구소의 첨단 기술, 과학의 중국 아카데미에 의해 승인 되는 프로토콜 (SIAT-IRB-160520-YYS-FXJ-A0232)에 엄격 하 게 따 랐 다.
1. 태 반 Chondroitin 황산 염 A 대상 지질 고분자 나노 입자의 합성
<ol><li>MTX 및 ICG 로드 지질 고분자 나노 입자를 합성 (MNPs와 INPs 각각) plCSA BP 활용 된 나노 입자 (plCSA-MNPs와 plCSA INPs)에 설명 된 대로 자세하게 다른18.</li>
</ol>2. vivo 형광 이미징
<ol><li>
임신 쥐의 준비
	<ol>
<li>한 장에 동일한 긴장의 비옥한 남성 여성 CD-1 마우스 (8-12 주) 장소 (남성: 여성 = 1:2) 확인 하 고 오후에는 질 플러그 다음 아침. 질 플러그 관찰 되는 경우 배아 하루 0.5 (E0.5)으로 마우스를 정의 합니다.</li>
		<li>집 임신 마우스 혼자 어두운 14 h 빛/10 h 동물 병원 체 없는 방에 주기을 E14.5까지 음식과 물에 대 한 무료 액세스를 제공 합니다.</li>
	</ol>
</li>
	<li>
나노 입자의 정 맥 주입
	<ol>
<li>절차, 전에 0.22 μ m 주사기 필터를 통해은 나노 여과 의해 소독. 수량 및 나노 사출의 볼륨을 결정 하기 위해 E11.5에서 임신 마우스 무게. 참고: 나노 사출 볼륨 1% 미만 (볼륨/중량) 임신 마우스의 몸 무게의 이어야 한다. 예를 들어 나노 사출 볼륨 25 g 마우스에 0.25 mL 미만 이어야 한다.</li>
		<li>꼬리 정 맥 팽창가 열 패드와 함께 5-10 분에 대 한 꼬리를 따뜻한.</li>
		<li>28 g 인슐린 주사기에 주입 하기 전에 INPs 또는 plCSA INPs 발음.</li>
		<li>임신 마우스를 꼬리 정 맥에 대 한 액세스를 허용 하는 동안 마우스를 멈출 지주 장치 전송. 꼬리는 알코올 면봉으로 청소 합니다. 다음 꼬리 정 맥에 주사기를 삽입 합니다. 천천히 주입 INPs 또는 plCSA-INPs (5 mg/kg ICG 해당)도 압력에 5-10 s. 참고: 물집에 나타나면 꼬리 때문에이 결과 바늘 임을 나타냅니다 하지 정 맥 주입을 중지 합니다. 주사기는 질병 전송 최소화 하기 위해 마우스 및 교차 오염을 사이 공유 되지 않을.</li>
		<li>사출 시간을 기록 합니다. 한편,는 일반적으로 30-60 s 출혈 정지까지 사출 사이트에 부드러운 압력을 적용 됩니다.</li>
	</ol>
</li>
	<li>
Vivo에서 화상 진 찰
	<ol>
<li>주입 후 30 분 임신 쥐에서 vivo 형광 이미징 시스템을 사용 하 여 이미지.</li>
		<li>Anesthetize 1.0 L/min와 isoflurane 마 취 단위의 관련된 상공에 2-4%의 산소 흐름 속도와 임신 쥐 고 느린에 의해 전체 마 취와 일반 호흡을 확인 합니다. 그런 다음 이미징 챔버로 이동 합니다. 부정사 위치에 동물 유지 이미징 실로 마 취 임신 마우스를 놓습니다.</li>
		<li>입과 마 취를 유지 하기 위해 1.0 L/min의 산소 흐름 속도 1-2 %isoflurane 흡입 수 있도록 코 코 콘 장소.</li>
		<li>ICG 형광 신호를 이미지를 2D 형광 및 사진 매개 변수를 선택 합니다. 설정 자동 으로 노출과에 여기/방출 파장 710/820 nm.</li>
		<li>이미징 절차의 끝에, 마 취, 막으려고 isoflurane 유입 해제 하 고 신중 하 게 그들의 감 금 소를 임신 쥐를 반환.</li>
		<li>나노 사출 후 48 h anesthetize isofluorane와 임신 쥐 그리고 자 궁 탈 댐 희생. 태아와 반응과 Graefe 집게, Graefe 조직 집게를 사용 하 고 해 부가 위를 수집 합니다.</li>
		<li>반응과 태아 이미징 실과 단계 2.3.4에서에서 설명 하는 방법을 사용 하 여 이미지에 놓습니다.</li>
	</ol>
</li>
</ol>3. HFUS 배아 발달의 평가
<ol><li>
동물 모델
	<ol>
<li>얻기 및 단계 2.1에에서 설명 된 대로 임신 쥐를 준비.</li>
		<li>HFUS를 사용 하 여 E 6.5 (3.2 및 3.3.3 프로토콜)에서 이미지 임신 쥐. 첫째, 하루 E6.5에 배아를 시각화 하 여 임신을 확인 하 고 다음 세 그룹으로 임신 쥐를 무작위로 할당: MNP 그룹, plCSA MNP 그룹 및 인산 염 버퍼 식 염 수 (PBS) 그룹.</li>
		<li>주사 PBS, MNPs 또는 plCSA-MNPs (1 mg/kg MTX 상응) 임신 쥐의 꼬리 정 맥으로 매일 2.2 단계에서 설명한 대로 E6.5에서 시작.</li>
	</ol>
</li>
	<li>
이미징에 대 한 준비
	<ol>
<li>나노 입자, 주사 후 24 h 이미지 이미징 시스템 HFUS를 사용 하 여 임신 쥐.</li>
		<li>2.3.2 단계에서 설명한 대로 임신 쥐를 anesthetize. 이미징 플랫폼의 통합된 온도 제어를 켜고, 37-42 ° c 플랫폼을 예 열 테이프를 사용 하 여 플랫폼에 부정사 위치에 임신 쥐를 보호 합니다.</li>
		<li>장소 코 콘 주 둥이 통해 마 취 단위에 연결. 1.0 L/min 꾸준한 마 취를 유지 하는 산소 유량과 2 %isoflurane 적용 됩니다.</li>
		<li>화학적 depilatory 크림을 사용 하 여 복 부에서 머리를 제거 합니다. 물에 젖은 거 즈로 깨끗이 잔여 크림을 닦 고 젤 커플링 음향 복 코트.</li>
	</ol>
</li>
	<li>
영상 절차
	<ol>
<li>기계 팔에서 40 MHz 변환기를 놓습니다.</li>
		<li>초점 영역에서 관심 영역으로 태아와 태 반에의 경도 이미지를 변환기 위치를 조정 합니다.</li>
		<li>
B 모드 영상과 분석 참고: 영화 1<ol>
<li>B 모드 버튼을 클릭 하 고 태아와 태 반 보기에 올 때까지 복 부에 변환기를 낮은. 시작/정지 영상, 씨 네 저장 을 누릅니다을 씨 네 루프 저장 프레임 저장 프레임 이미지를 저장 하 게 스캔/동결 을 누릅니다.</li>
			<li>임신 성 골목 길이 (GS), 태아 크라운 둔 부 길이 (CRL), biparietal 직경 (BPD), 복 부 둘레 (AC), placental 직경 (PD)와 태 반 두께 (PT) 분석 측정 버튼을 클릭 합니다.</li>
		</ol>
</li>
		<li>
PW 도플러 영상 및 분석 참고: 영화 1<ol>
<li>동일을 사용 하 여 프로젝션 스캔, 비밀 번호 버튼을 클릭, 샘플링 볼륨 상자 탯 줄 동맥의 중심에 놓고 스캔/동결 이미징 시작을 누릅니다. 탯 줄 동맥 이미지 수집 하 씨 네 저장 을 클릭 합니다.</li>
			<li>탯 줄 동맥 피크 속도 (UA)를 계산 하기 위해 측정 버튼을 클릭 합니다.</li>
		</ol>
</li>
		<li>
색 도플러 모드 영상 및 분석
		<ol>
<li>동일을 사용 하 여 프로젝션 스캔 컬러 버튼을 클릭을 태아 심장의 이미지를 변환기 위치를 조정. 스캔/동결 이미징 및 씨 네 이미지 수집 저장 시작을 누릅니다.</li>
			<li>태아의 심장 박동 (인사)을 계산 하려면 측정값 단추를 클릭 합니다.</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. HPLC 분석
<ol><li>
조직 준비
	<ol>
<li>늦은 임신에서 MNPs 또는 plCSA-MNPs (1 mg/kg MTX 동등 물)의 단 하나의 복용량을 임신 쥐를 주입 (예., E14.5) 단계 3.1.3에서에서 설명한 대로.</li>
		<li>24 시간 후 240 μ g/몸 무게 (g)에서 avertin의 복 주사 하 여 쥐를 anesthetize. 생쥐는 완전히 마 취 확인 하 발 핀치에 응답을 확인 합니다.</li>
		<li>스프레이 75% 에탄올과 가슴 지역입니다. 심장 관류 (잘라 오른쪽 심 방 및 좌 심 실을 통해 perfuse) 수행 세부19,20 언바운드 나노 입자를 제거 하는 10 분 동안 차가운 0.9% 염 분의 50 mL에에서 앞에서 설명한.</li>
		<li>댐 안락사 태아와 반응과 Graefe 집게가 위, 그리고 Graefe 조직 겸 자, 해 부를 사용 하 여 수집 하 제왕 섹션을 수행 하 고 조직 분석 전에-80 ° C에서 저장 합니다.</li>
		<li>균질 솔루션 (10%과 염소 산)을 준비 하 고 얼음에 계속. 조직의 약 200 mg을 수집 하 고 각 샘플을 균질 솔루션의 500 μ를 추가. 균질 샘플 30 s, 그리고 반복에 대 한 최고 속도 균질 화기를 사용 하 여이 절차를 두 번.</li>
		<li>4 ° c.에 20 분 14000 × g에서 샘플을 원심 HPLC 유리병에 결과 액체를 전송 및 필터 주사기 0.45 μ m 통해 상쾌한 (약 300 μ)을 필터링 합니다. 샘플 튜브를 주입 autosampler 트레이 넣으십시오.</li>
	</ol>
</li>
	<li>
표준의 준비
	<ol>
<li>다음 솔루션 모바일 단계에 대 한 준비: 40 mM 칼륨 인산 염기 (pH 4.5)와 이기 (88:12, v/v). 0.45 μ m 기 공 크기 주사기 필터를 통해 솔루션을 필터링 하 고 깨끗 한 HPLC 저수지 병 결과 액체를 전송. 참고: 0.1 M 인산으로 pH를 조정 합니다. 15 분에 대 한 초음파 진동을 사용 하 여 사용 하는 모바일 단계 때마다 이전 드.</li>
		<li>1.5 mL 원심 분리기 관으로 MTX의 10 밀리 그램 무게. 1 M 수산화 나트륨의 1 mL를 추가 합니다.</li>
		<li>소용돌이 MTX까지 고속에서 완전히 녹 인 다. 참고:이 기본 주식 이며 몇 달 동안-20 ° C에 저장 될 수 있습니다.</li>
		<li>보조 MTX 주식 (500 μ g/mL)를 만들려면 50 μ 모바일 단계의 950 μ에 기본 주식의 희석. 참고: 사용까지 얼음에 저장 하 고 매일 신선한 준비. 사용 하 여 모바일 단계 표준의 준비에 대 한 샘플 주입 후 서로 다른 솔루션을 혼합의 결과로 봉우리를 피하기 위해 중요 하다.</li>
		<li>(표 1) 표준을 만들고 희석 추가 확인 합니다. 얼음에 기준을 저장 하 고 매일 신선한 준비. 표준 직렬 실험 샘플으로 실행 합니다.</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>번호</td>
			<td>최종 농도 (μ g/mL)</td>
			<td>500 μ g/mL, μ</td>
			<td>모바일 phase(μL)</td>
		</tr>
<tr>
<td>1</td>
			<td>0.5</td>
			<td>1</td>
			<td>999</td>
		</tr>
<tr>
<td>2</td>
			<td>1</td>
			<td>2</td>
			<td>998</td>
		</tr>
<tr>
<td>3</td>
			<td>2.5</td>
			<td>5</td>
			<td>995</td>
		</tr>
<tr>
<td>4</td>
			<td>10</td>
			<td>20</td>
			<td>980</td>
		</tr>
<tr>
<td>5</td>
			<td>25</td>
			<td>50</td>
			<td>950</td>
		</tr>
<tr>
<td>6</td>
			<td>50</td>
			<td>100</td>
			<td>900</td>
		</tr>
<tr>
<td>7</td>
			<td>100</td>
			<td>200</td>
			<td>800</td>
		</tr>
</tbody></table>표 1입니다. 표준 곡선의 MTX에 대 한 준비. MTX 표준 솔루션의 최종 농도 0.5-100에서 μ g/mL.
<ol start="3"><li>
HPLC 계측 및 작업 매개 변수 참고: 샘플 용 펌프, UV spectrophotometric 검출기를 장착 하는 HPLC 시스템에 분석 되었다 (313 nm), 및 C18 열 (250 × 4.6 m m, 5 μ m 입자 크기).<ol>
<li>시스템에서 공기를 제거 하는 HPLC degasser를 켭니다. 초기 소음을 줄이기 위해 30 분 모바일 단계와 열 평형 흐름을 켭니다.</li>
		<li>열의 온도 25 ° C를 설정 하 고 1 mL/min의 유량에 20 μ 샘플 볼륨을 주입 실행 방법 분석 시작을 클릭 합니다.</li>
		<li>실행 완료 되 면 수동으로 HPLC 급 이기에 모바일 단계를 변경 합니다. 시스템을 보호 하기 위해 약 15 분 동안 실행 합니다. 참고: 권장된 실행 시간을 다음이 단계를 수행 하려면 실패 손상에서 열 될 수 있습니다.</li>
		<li>정량 분석을 위한 HPLC 시스템 소프트웨어를 사용 하 여 관심사의 표준 MTX 봉우리 아래 영역을 계산 합니다.</li>
	</ol>
</li>
</ol>

<ol>
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X.F.와 B.Z.는 민 태 반 관련 약물 전달 방법을 포함 하 여 특허 출원 PCT/CN2017/108646 제출 및 그 응용 프로그램에 대 한 발명가. 다른 모든 저자 들은 아무 경쟁 관심사를 선언 합니다.

이 원고에서 우리 plCSA BP-기반 나노 입자 약물의 태 반 배달 대상에 대 한 효율적인 도구 인지 결정 하기 위한 3-방법 시스템 개요. Vivo에서 이미징 적외선 형광 ICG 신호를 모니터링 하는 데 사용 plCSA BP. 사용의 placental 대상 특이성을 확인 HFUS 및 HPLC, 우리 plCSA BP 활용 된 나노 입자 수에 MTX 제공 효율적으로 시연 합니다 태 반 세포, 태아에 게 하지입니다.
Vivo에서 형...

사전 eclampsia, 임신 손실, placental 속보 및 작은 임신 연령 (SGA)를 포함 하 여 중재 하는 태 반 임신 합병증은 일반적이 고 실질적인 태아와 산 모 병 적 상태와 사망률1,2,에 3, 그리고 거의 약4,5장애 임신 치료에 효과적인 것으로 입증 되었습니다. 임신 중 더 선택적이 고 안전한 태 반 대상 약물 전달에 대 한 전략의 개발 현대 약물 치료에 도전 남아 있습니다.
최근 몇 년 동안, 여러 보고서 태 타겟팅 도구로 펩 티 드 또는 항 체 나노 입자 코팅에 의해 uteroplacental 조직에 약물의 타겟된 배달에 집중 했다. 이러한 안티 표 피 성장 인자 수용 체 (EGFR)6 항 체, 등 종양 유도 펩 티 드 (CGKRK 및 iRGD)7, 태 반 대상 펩 티 드8, placental 맥 관 구조 대상 펩 티 드9 에 대 한 항 체는 옥 시 토 신 수용 체10.
여기, 합성 placental chondroitin 황산 염 A 바인딩 펩 티 드 (plCSA-BP) 태11나노 입자와 그들의 마약 페이로드의 대상된 배달에 사용할 수 있습니다 설명 합니다. PlCSA BP-기반 나노 입자는 placental trophoblast 대상 때문에 메서드를 대상으로 보고 uteroplacental를 보완.
비-침략 적 방법으로 이미징 vivo에서 쥐12, 태 반 관련 유전자 발현을 모니터링 하는 데 사용 되었습니다 그리고 indocyanine 그린 (ICG) 나노 입자 형광 이미징 시스템13를 사용 하 여 추적을 널리 이용 되는 , 1415. 따라서, 우리는 plCSA BP 활용 된 나노 로드 ICG (plCSA-INPs) 형광 이미징 임신 쥐에 plCSA INP 분포를 시각화 하 정 맥 주입. 우리는 다음 정 맥 methotrexate (MTX) 주사-임신 쥐에 plCSA-NPs를 로드. 높은 주파수 초음파 (HFUS), 다른 비-침략 적, 실시간 이미징 도구16,17 쥐에 태아 및 태 반 개발을 모니터링 하는 데 사용 되었다. 마지막으로, 우리 반응과 태아에 MTX 분포를 정량화 고성능 액체 크로마토그래피 (HPLC) 사용.
이 프로토콜에서 우리가 자세히 설명 plCSA 혈압 유도 nanocarriers 태 대상 약물 전달의 효율성을 평가 하는 데 사용 하는 3 방법 시스템.

<table><tbody><tr><td>CD-1 mice</td><td>Beijing Vital River</td><td>201</td><td>Female (8-12 week)</td></tr><tr><td>Insulin syringe</td><td>BD</td><td>328421</td><td>for IV injection</td></tr><tr><td>Ethanol absolute</td><td>Sinopharm Chemical</td><td>10009218</td><td>for nanoparticles synthesis</td></tr><tr><td>Soybean lecithin</td><td>Avanti Polar Lipids</td><td>441601</td><td>for nanoparticles synthesis</td></tr><tr><td>DSPE-PEG-COOH</td><td>Avanti Polar Lipids</td><td>880125</td><td>for nanoparticles synthesis</td></tr><tr><td>PLGA</td><td>Sigma-Aldrich</td><td>719897</td><td>for nanoparticles synthesis</td></tr><tr><td>Ultrasonic processor</td><td>Sonics</td><td>VCX130</td><td>for nanoparticles synthesis</td></tr><tr><td>Methotrexate (MTX)</td><td>Sigma-Aldrich</td><td>V900324</td><td>for nanoparticles synthesis</td></tr><tr><td>Indocyanine green (ICG)</td><td>Sigma-Aldrich</td><td>1340009</td><td>for in vivo imaging</td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Hyclone</td><td>SH30028.01</td><td></td></tr><tr><td>IVIS spectrum instrument</td><td>Perkin Elmer</td><td></td><td>for in vivo imaging</td></tr><tr><td>Ultrasound transmission gel</td><td>Guanggong</td><td>ZC4252418</td><td>for ultrasound imaging</td></tr><tr><td>Isoflurane</td><td>Lunan Pharmaceutical</td><td>I0040</td><td>for maintain the anesthesia</td></tr><tr><td>Depilatory cream</td><td>Nair</td><td>TMG001</td><td>for removing fur</td></tr><tr><td>40 MHz transducer</td><td>VisualSonics</td><td>MS550S</td><td>for ultrasound imaging</td></tr><tr><td>High-frequency ultrasound imaging system</td><td>VisualSonics</td><td>Vevo2100</td><td>for ultrasound imaging</td></tr><tr><td>Avertin</td><td>Sigma-Aldrich</td><td>T48402</td><td>for anesthesia</td></tr><tr><td>Syringe pump</td><td>Mindray</td><td>SK-500III</td><td>forcardiac perfusion</td></tr><tr><td>0.9% saline solution</td><td>Meilunbio</td><td>MA0083</td><td>forcardiac perfusion</td></tr><tr><td>1.5 mL Polypropylene tubes</td><td>AXYGEN</td><td>MCT-150-C</td><td></td></tr><tr><td>-80 &amp;deg;C freezer</td><td>Thermo Fisher Scientific</td><td>88600V</td><td></td></tr><tr><td>Centriguge</td><td>Cence</td><td>H1650R</td><td></td></tr><tr><td>Perchloric acid</td><td>Sigma-Aldrich</td><td>311421</td><td>for precipitating protein</td></tr><tr><td>Homogenizer</td><td>SCIENTZ</td><td>SCIENTZ-48</td><td>for homogenizing tissue</td></tr><tr><td>Syringe filter (0.45 &amp;mu;m)</td><td>Millipore</td><td>SLHV033RS01</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sinopharm Chemical</td><td>10019763</td><td>for solving MTX</td></tr><tr><td>HPLC vials</td><td>Waters</td><td>670650620</td><td>for HPLC</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sinopharm Chemical</td><td>20032117</td><td>for HPLC</td></tr><tr><td>Acetonitrile</td><td>JKchemical</td><td>932537</td><td>for HPLC</td></tr><tr><td>C18 column</td><td>Waters</td><td>186003966</td><td>for HPLC</td></tr><tr><td>HPLC system</td><td>Shimadzu</td><td></td><td>for HPLC</td></tr></tbody></table>

comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods

더 효과적인 치료 태 반 관련 임신 합병증에 대 한 현재 존재 하 고 도전 남아 태아와 모성의 부작용을 최소화 하면서 태 반에 약물의 타겟된 배달에 대 한 전략을 개발. 타겟된 나노 캐리어 placental 질환을 치료 하는 새로운 기회를 제공 합니다. 우리는 최근에 합성 placental chondroitin 황산 염 A 바인딩 펩 티 드 (plCSA-BP) 태 반에 약물을 전달 하기 위해 나노 가이드 사용 될 수 보여 주었다. 이 프로토콜에서 설명 자세히 plCSA-혈압 사용 세 별도 메서드를 사용 하 여 태 반에 약물 전달의 효율성을 평가 하기 위한 시스템: vivo에서 이미징, 높은-주파수 초음파 (HFUS), 및 높은 성능 액체 착 색 인쇄기 (HPLC)입니다. Vivo에서 를 사용 하 여 이미징, plCSA BP-기반 나노 했다 시각에 살아있는 동물의 반응과 HFUS 및 HPLC는 plCSA BP 활용 된 나노 효율적이 고 구체적으로 전달 토트 태 반에 시연 하는 동안. 따라서, 이러한 방법의 조합은 태 반에 약물의 타겟된 배달 및 여러 가지 임신 합병증에 대 한 새로운 치료 전략의 개발을 위한 효과적인 도구로 사용할 수 있습니다.

이 원고, plCSA BP 활용 된 나노 로드 MTX (plCSA-MNPs) 또는 ICG (plCSA-INPs) 했다 임신 쥐에 정 맥 주입 됩니다. Vivo에서 영상 plCSA INP 주입 후 자 궁 30 분의 지역에서 강한 ICG 신호를 밝혔다. INPs 했다 주로 간, 비장 지역 (그림 1A) 지역화 됩니다. PlCSA-INP 주사 후 48 h, 임신 쥐 ICG 신호 없는 신호와 태 반에만 태아 (그림 1B)에 감지 했다 ?...

Watch this Scientific Journal Video about Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods at JoVE.com

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

우리는 안전 및 태 반 대상 약물 전달의 효과 평가 하기 위해 세 가지 방법을 사용 하는 시스템을 설명: vivo에서 이미징 나노 축적, 태 반 및 태아 개발을 모니터링 하 고 주파수 초음파를 모니터링 하 그리고 조직에 약물 전달 척도를 HPLC.

3 보완 방법을 사용 하 여 태 반 대상 약물 전달의 안전과 효율성의 종합 평가

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

comprehensive-evaluation-effectiveness-safety-placenta-targeted-drug

Research

JoVE Journal

Bioengineering

9.6K 조회수.  Chinese Academy of Sciences. 우리는 안전 및 태 반 대상 약물 전달의 효과 평가 하기 위해 세 가지 방법을 사용 하는 시스템을 설명: vivo에서 이미징 나노 축적, 태 반 및 태아 개발을 모니터링 하 고 주파수 초음파를 모니터링 하 그리고 조직에 약물 전달 척도를 HPLC.

비디오: Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

갓 고립 된 인간의 산모 Cytotrophoblasts에 siRNA를 형질 및 EMSA 분석

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

연구

유전학

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic 트윈 Placentas의 혈관 매핑을위한 가이드 와이어 원호 Catheterization과 컬러 염료 사출

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

의학

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

유색 염료를 사용하여 Monochorionic 태반의 혈관 Anastomoses의 정확하고 간단한 평가

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

합성 및 Placental 콘의 황산 (plCSA)-지질 타겟팅-고분자 나노 입자

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

암 연구학

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

태반의 맞은 편에 생체이 물과 나노 물질의 전송 속도의 결정을 사용하여<em&gt; 생체</em&gt; 인간 태반 관류 모델

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

생체공학

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

모델 및 세포 장벽에 걸쳐 약물 전달 시스템의 전송을 평가하는 방법

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

Mouse In Vivo Placental 표적 CRISPR 조작

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

설치류 태 반의 전 생체 관류

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

발생학

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

액정 나노 입자 캐리어의 소수성화물 캡슐화의 대상 플라즈마 멤브레인 배달

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

인간 태반 생리학의 통합 연구에 대한 4 혈관 샘플링 접근법<em&gt; In Vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

3 보완 방법을 사용 하 여 태 반 대상 약물 전달의 안전과 효율성의 종합 평가

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